Calculating per-cell intensity over time in .nd2 files for calcium imaging

Hi everyone,

I started using CellProfiler a couple hours ago, and I could use some help with my first hamfisted attempt at making a pipeline.

I have time-lapse images of neurons stained with a calcium indicator, saved as .nd2 files (I can also do .tif files with frames if that works better). I would like to have a spreadsheet showing each cell’s intensity in each frame, so I can plot them and figure out how many calcium events each cell had during the imaging session.

There are some modules that are obviously related to what I need to do (IdentifyPrimaryObjects, MeasureObjectIntensity, ExportToSpreadsheet) but when I run the pipeline I made from them, it only seems to measure one frame of the image. I think it understands that the file is actually a stack, since I have the option to analyze as 2-D or 3-D and I can make a maximum intensity projection, but there’s only one frame’s worth of data in the output spreadsheet. How can I get it to do all of the frames?

I’m sure this must have been asked before, but when I search I’m mainly turning up questions about processing movies where each frame is a separate file.
test.cpproj (417.1 KB)
Results_Cells.csv (153.9 KB)
(Edit: it looks like my tiff file is too big to attach–let me know if it would help to provide a more detailed description or a couple example frames)

Hi @masher,

It’s difficult to supply precise instructions without having the image file itself to look at, but checking the pipeline you uploaded it looks like you just need to tweak some settings for identifying the images. I’d suggest that you try enabling metadata extraction in the Metadata module, and choose the method “Extract from image file headers”. This should then allow CellProfiler to pull out each frame of the time series.

Hope that helps!

That fixed it, thank you!